Detection of synthetic cannabinoids

ABSTRACT

The invention describes methods and kits for detecting and determining current and future synthetic cannabinoids from the JWH and CP families. Unique antibodies derived from novel immunogens enable said methods and kits.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of, and claims priority to,U.S. patent application Ser. No. 13/332,042, filed Dec. 20, 2011, nowU.S. Pat. No. 8,906,633, which claims priority to GB Patent ApplicationsNo. 1102544.2, filed Feb. 14, 2011, and No. 1110425.4, filed Jun. 21,2011, all of which applications are incorporated herein by reference intheir entireties.

BACKGROUND

The increasing rise in the use of stealth drugs (novel synthetic drugsthat were previously or remain analytically/structurally uncharacterisedand unclassified by government institutions) is exemplified by syntheticcannabinoid products which incorporate JWH-018 and/or CP 47,497 as theactive ingredient. Stealth synthetic cannabinoid (SSC) drugmanufacturers can base their choice of active molecular target onscientific literature studies that address the therapeutic potential ofCB1 (the CNS cannabinoid receptor) agonists and antagonists. Byincorporating novel, analytically uncharacterised compounds with highCB1 receptor affinity into herbal mixtures (packaged under such names asSpice, Yucatan Fire) the manufacturers are able to legally target drugconsumers clandestinely by promoting the material as herbaltherapeutics. A problem for governments and drug enforcement agencies isthat even after identifying and banning a new synthetic cannabinoid, themanufacturers can rapidly react to the banning by incorporating adifferent active analogue into the same or a different herbal product;targeted minor changes in the molecular structure of the known activecompound can preserve receptor activity but often produces a moleculewhose GC-MS/LC-MS (the commonly applied detection techniques) profile iscompletely different from the original active molecule. Hence the newactive molecule initially remains unidentified and a further resourceintensive and costly chemical analytical study to enable structuralcharacterisation is required. The main active ingredients highlighted inSSC products to date are JWH-018, CP 47,497 and JWH-073 (Uchiyama et al.2010; Hudson et al. 2010; Dresen et al. 2010). Initial studies of themetabolism of JWH and CP compounds have highlighted metabolic processessimilar to tetrahydrocannabinol (THC) metabolism, namely ring and alkylsubstituent hydroxylation, carboxylation and glucuronidation. Asdescribed herein, unless otherwise stated, JWH refers to moleculescomprising structure I which are CB1-active or metabolites of theCB1-active parent, in which the indole ring system is present as a fusedheterobicyclic, i.e., it is not part of, for example, a fusedheterotricyclic ring system. Y can be hydrogen or a substituted orunsubstituted alkyl group such as butyl, pentyl or2-(morpholin-4-yl)ethyl, while R is a carbon atom which may be part of afused or unfused, substituted or unsubstituted aromatic ring or asubstituted or unsubstituted alkyl, alkenyl or alkynyl chain optionallyattached to a fused or unfused, substituted or unsubstituted aromaticring, but is usually a substituted or unsubstituted naphthyl ring.

CP refers to synthetic cannabinoid molecules comprising the unfusedbicyclic structure II, in which X is either ethyl, n-propyl or n-butylas well as metabolites thereof.

Indolyl, naphthyl, carboxyalkyl, N-dealkylated and N-alkyl mono-, di-,and tri-hydroxylated metabolites, as well as their glucuronidatedconjugates are reported JWH-018 metabolites (Sobolevsky et al. 2010;Kraemer et al. 2008). Moller et al. (2010) highlighted the samemetabolites as Sobolevsky et al. (2010), with the monohydroxylatedN-alkyl chain being the most abundant phase I metabolite; Wintermeyer etal. (2010) conducted an in vitro study that largely confirmed previousfindings. Herbal therapeutics have been analysed using solventextraction, pre-derivatisation and finally GC-MS analysis in SIM mode(Rana et al. 2010). This method is inadequate for the detection offuture and ‘current’ JWH and CP SSCs (it is conceivable that ‘current’herbal therapeutics, as well as JWH-018 and CP 47,497, incorporate JWHand CP SSCs that are not yet characterised), requires samplepre-derivatisation, specialist staff for its implementation andexpensive equipment. In order to address the problem associated with thecheap and rapid detection of known JWH and CP molecules and theirmetabolites and/or future and associated metabolites based on the JWHand CP drug families, the Inventors devised a novel method based onnovel antibodies raised from novel immunogens. The antibodies underpinan effective analytical and economic solution to the detection andquantification of current and future JWH and CP CB1-active molecules inin vitro patient samples and herbal therapeutics.

SUMMARY OF THE INVENTION

The invention describes a rapid and practical method for the detectionand determination of known and/or stealth synthetic cannabinoids basedon the JWH and CP drug families. Kits and their use for JWH and CP SSCdetection and determination in herbal therapeutics and in vitro patientsamples are also described. The invention is underpinned by novelimmunogens and antibodies which enable said methods, kits andapplications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the synthesis of Hapten-A.

FIG. 2 is a diagram showing the synthesis of Hapten-B.

FIG. 3 is a diagram showing the synthesis of Hapten-C.

FIG. 4 is a diagram showing the synthesis of Hapten-D.

FIG. 5 is an illustration of Immunogens I, II and III.

FIG. 6 is an illustration of representative molecules of the JWH family.

FIG. 7 is an illustration of identified and hypothesised metabolites ofJWH (n=1-2). The hydroxylated metabolites are potentially furthermetabolised to the corresponding glucuronide(s).

FIG. 8 is an illustration of CB1-active molecules of the CP family(available from Cayman Chemical company, 1180 East Ellsworth Road, AnnArbor, Mich. 48108, USA).

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the invention is one or more immunogens possessing thefollowing structures

in which the accm is an antigenicity conferring carrier material; thecrosslinker is a functionalised linking group joining the accm to theremainder of the molecule.

By functionalised it is meant the crosslinker incorporates atoms thatenable it to bond to both the accm and the JWH or CP moiety, forming abridging group. In structure (e) the crosslinker forms either a singleor double bond to the cyclohexyl ring, in structure (c) the crosslinkerextends from the 4, 5, 6 or 7-position of the indole ring and instructure (d) the crosslinker extends from the 2, 3, 4, 5, 6, 7, or8-position of the naphthyl ring. The crosslinker concept is well knownto the person skilled in immunogen synthesis. For the current invention,when conjugating the hapten to the accm to form the immunogen, thenature and length of the crosslinker follows standard methods in orderto optimise hapten epitopic recognition by the antibody. This entails acrosslinker of low immunogenicity and a chain length preferably of nogreater than about ten atoms, most preferably no greater than six atoms.

Preferably for structure (a) the crosslinker is —(CO)_(n)-D-Y— and wheren=0 or 1, and D is a C₁₋₁₀, preferably a C₁₋₅ substituted orunsubstituted straight chain alkylene or arylene moiety and Y, which isattached to the accm, is selected from groups such as carbonyl, amino,thiol, maleimide, isocyanato, isothiocyanato, aldehyde, diazo anddithiopyridyl. Y is preferably carbonyl or amino.

Preferably for structures (b), (c), (d), (f) and (h) the crosslinker is-(A)_(n)-D-Y— where A=O, —N(R)—, S, —S(O)— (sulphoxide) or —S(O)₂—(sulphonyl) and R=H or C₁₋₅ alkyl, n=0 or 1 and D is a C₁₋₁₀, preferablya C₁₋₅ substituted or unsubstituted straight chain alkylene or arylenemoiety and Y, which is attached to the accm, is selected from groupssuch as carbonyl, amino, thiol, maleimide, isocyanato, isothiocyanato,aldehyde, diazo and dithiopyridyl. Y is preferably carbonyl or amino.

Preferably for structure (e) m=1-3, the crosslinker is either-(L)_(p)-M-Q- or ═N—O-M-Q- in which Q, attached to the accm, is eithercarbonyl or amino, M is a C₁₋₁₀, preferably a C₁₋₅ substituted orunsubstituted straight chain alkylene or arylene moiety, p=0 or 1 and Lis O, NH, S, ester, thioester, or amide.

Preferably for structure (g) m=1-3, the crosslinker is -(L)_(p)-M-N— inwhich N, attached to the accm, is either carbonyl or amino, M is aC₁₋₁₀, preferably a C₁₋₅ substituted or unsubstituted straight chainalkylene or arylene moiety, p=0 or 1 and L is O, NH, S, ester,thioester, or amide.

Preferred immunogens correspond to structures (a) and (c). Thecrosslinker of structure (c) of Group I is preferably attached to the5-indole position. The crosslinker of structure (d) of Group I ispreferably attached to the 4-naphthyl position. It has been found thatimmunogens of the invention raise antibodies that are able to bind toseveral JWH molecules and metabolites. The skilled person is aware thatfor these antibodies to recognize JWH and CP molecules they must bind toparticular structures or epitopes of the hapten (in this context thehapten being that part of the immunogen that is not the crosslinker oraccm); the epitopes are often distinct groups incorporating functionalgroups. For example, with reference to the JWH immunogen of structure(a) of Group I, the epitope recognized by the antibody will be all orpart of the 3-(1-naphthoyl)-1H-indole moiety, and for all immunogen ofstructure (b) of Group I the epitope recognized by the antibody will beall or part of the N-pentyl-3-carbonyl-1H-indole moiety. The accm can beany material that makes all or part of the hapten susceptible toantibody recognition and binding. For example the accm can be a protein,a protein fragment, a synthetic polypeptide or a semi-syntheticpolypeptide. Especially preferred immunogens of the invention correspondto structures (a) and (c) of Group I in which structure (a) hasY=carbonyl, n=0 and D=pentylene and structure (c) has Y=carbonyl, A=O,n=1 and D=methylene.

A further aspect of the invention is an antibody raised against animmunogen of structure (a), (b) or (c) of Group I, that is able to bindto molecules of the JWH family and their metabolites that comprisestructure I. The term ‘able to bind to’, as used herein, does not implythat the antibodies have a choice of whether or not to bind to the JWHmolecules, but that under standard immunoassay conditions the antibodieswill bind to the JWH or CP molecules. The antibodies are preferablyraised against immunogens of structure (a) or (c) of Group I, theantibodies being able to bind to several molecules and metabolites ofthe JWH family, including JWH-018 and its N-alkyl hydroxylatedmetabolites. It is especially preferred that the antibodies are raisedfrom structure (a) of Group I in which Y=carbonyl, n=0 and D=pentyleneand structure (c) of Group I in which Y=carbonyl, A=O, n=1 andD=methylene, the antibodies able to bind to several molecules andmetabolites of the JWH family, including JWH-018 and its dealkylated,hydroxylated and carboxylated metabolites, JWH-073, JWH-081, JWH-200 andJWH-398. Preferably, the antibodies raised against immunogens ofstructure (a) or (c) of Group I are also able to bind to one or moremetabolites of JWH-018 identified as M1-M5 in FIG. 7, namely JWH-018N-pentanoic acid metabolite (M1), JWH-018 5-hydroxyindole metabolite(M2), JWH-018 4-hydroxyindole metabolite (M3), JWH-018N-(5-hydroxypentyl) metabolite (M4) and JWH-018 6-hydroxyindolemetabolite (M5); and/or the JWH-018 metabolite JWH-018N-(4-hydroxypentyl) metabolite; and/or the 5-fluoropentyl derivative ofJWH-018, 1-(5-fluoropentyl)indol-3-yl(1-naphthyl)methanone; and/or theJWH-018 metabolite identified as JWH-250 in FIG. 6; and/or one or moreJWH-073 metabolites selected from JWH-073 N-(3-hydroxybutyl) metaboliteand JWH-073 N-(4-hydroxybutyl) metabolite. These compounds are availablefrom, for example, Cayman Chemical Company. The antibodies also have thepotential to bind to CB1-active derivatives of JWH molecules that couldrepresent future generations of SSCs.

Optionally, the antibodies may have broad cross-reactivity across theJWH family and metabolites. For example, without intending to limit theinvention thereto, antibodies raised to Immunogen I (structure (a) ofGroup I) may be specific to JWH-018 N-(5-hydroxypentyl) metabolite (M4)and cross-reactive to JWH-073, JWH-200, JWH-018, JWH-018 N-pentanoicacid metabolite (M1), JWH-018 5-hydroxyindole metabolite (M2), JWH-0184-hydroxyindole metabolite (M3), JWH-018 6-hydroxyindole metabolite (M5)and 3-(1-naphthoyl)-1H-Indole. Similarly, antibodies raised to ImmunogenII (structure (c) of Group I) may be specific to JWH-018 6-hydroxyindolemetabolite (M5) and cross-reactive to JWH-073, JWH-200, JWH-398,JWH-018, JWH-018 N-pentanoic acid metabolite (M1), JWH-0185-hydroxyindole metabolite (M2), JWH-018 4-hydroxyindole metabolite (M3)and JWH-018 N-(5-hydroxypentyl) metabolite (M4).

Optionally, the antibodies raised to Immunogen I (structure (a) of GroupI) will be able to bind to an epitope of JWH-073, JWH-200, JWH-018,JWH-018 N-pentanoic acid metabolite (M1), JWH-018 5-hydroxyindolemetabolite (M2), JWH-018 4-hydroxyindole metabolite (M3), JWH-018N-(5-hydroxypentyl) metabolite (M4) and JWH-018 6-hydroxyindolemetabolite (M5). Optionally, the epitope will be all or part of3-(1-naphthoyl)-1H-Indole. Further optionally, the antibodies raised toImmunogen II (structure (c) of Group I) will be able to bind to anepitope of JWH-073, JWH-200, JWH-398, JWH-018, JWH-018 N-pentanoic acidmetabolite (M1), JWH-018 5-hydroxyindole metabolite (M2), JWH-0184-hydroxyindole metabolite (M3), JWH-018 N-(5-hydroxypentyl) metabolite(M4) and JWH-018 6-hydroxyindole metabolite (M5).

The invention also describes an antibody raised against an immunogen ofstructure (d), (e), (f) or (g) the antibody being able to bind tomolecules of the CP family, metabolites of CP molecules and future SSCmolecules comprising structure II. When used in reference to anantibody, the word specific in the context of the current inventionrefers to the analyte that is preferably bound by the antibody, asgauged by a suitable metric such as the IC50. Given the IC50 of variousanalytes their cross-reactivities can be calculated. The antibody caneither be a polyclonal or monoclonal antibody using well-known methods.If the polyclonal antibody possesses the required specificity andsensitivity, that is, it binds a single analyte within the detectionrange of the assay, development of a monoclonal antibody is unnecessary.Alternatively, a polyclonal or monoclonal antibody that binds to severalanalytes might be desirable; in the context of the current inventionantibodies that bind several analytes are preferred. One or moreantibodies of the invention can be incorporated into a kit for thedetection and determination of individual or multiple SSCs. The skilledperson in the immunodiagnostic field is aware of several alternativeimmunoassay formats that could incorporate the antibodies of theinvention either in solution or tethered (e.g. covalently bonded orelectrostatically ‘non-bonded’ through van der waal's forces) to a solidsubstrate such as beads, glass/plastic slides or ceramic chips (a chipdefined as a small, planar substrate). A preferred solid substrate ontowhich the antibodies of the invention are covalently bonded is a chip,preferably a ceramic chip; the word ‘biochip’ can be used to refer to achip with antibodies attached. Thus the invention also provides a solidsubstrate, preferably a biochip, comprising antibodies raised to animmunogen of one or more of structures (a), (b), (c), (d), (e), (f), (g)or (h), the antibodies being able to bind to an epitope of one or moremolecules of the JWH family and/or CP family and/or one or moremetabolites thereof. The antibodies of the invention can be used for thedetection or determination of a single SSC such as JWH-018, either asthe parent molecule or as a metabolite, but a preferred embodiment isthe use of one or more antibodies, preferably two or more antibodies, atleast one derived from Group I and one derived from Group II, for thedetection or determination of several SSCs and/or their metabolites fromthe JWH and CP families. The detection and determination criteria for aSSC using an immunoassay platform includes, as is well-known in the art,exceeding a pre-defined cut-off/concentration value or measuring thecalibrator equivalent value as derived from a calibrator curve (alsoreferred to as a standard curve).

Another aspect of the invention is a method of detecting or determiningsynthetic cannabinoids of the JWH and/or CP families and theirmetabolites in an in vitro sample of an individual or in a solutionderived from a substance suspected of containing synthetic cannabinoidscomprising: contacting the sample or solution with one or more detectingagents and one or more antibodies of the invention that bind tomolecules of the JWH family and/or one or more antibodies of theinvention that bind to molecules of the CP family, measuring thedetecting agents, and detecting or determining, by reference tocalibrators, the presence or concentration of a molecule or molecules ofthe JWH family and/or CP family. With reference to ‘detecting ordetermining’, ‘detecting’ means qualitatively analyzing for the presenceor absence of a substance, ‘determining’ means quantitatively analyzingfor the amount of a substance. The detecting agent is a small molecule,generally of similar structure to a molecule to be detected conjugatedto a labelling agent, that is able to bind to one of the antibodies ofthe invention. The labelling agent is selected from an enzyme, aluminescent substance, a radioactive substance, or a mixture thereof.Preferably, the labelling agent is an enzyme, preferably a peroxidase,most preferably horseradish peroxidase (HRP). Alternatively, oradditionally, the luminescent substance may be a bioluminescent,chemiluminescent or fluorescent material. For the purposes of theinvention, the patient sample to be used for in vitro analysis can behair or a peripheral biological fluid but is preferably whole blood,serum, plasma, or urine.

Preferably the synthetic cannabinoids to be detected or determined areone or more of JWH-018, JWH-073, JWH-200 and JWH-398 and the one or moreantibodies are derived from immunogens of structures (a) and (c) ofGroup I. When referring to the detection or determination of a JWH or CPmolecule, with or without a suffixed number attached to JWH and CP, themetabolite or metabolites are also inferred unless otherwise stated. Theimmunogen of structure (a) preferably has a crosslinker —X—Y— in which Yis carbonyl and is attached to the accm, and X is pentylene, and theimmunogen of structure (c) has a crosslinker —O—CH₂—C(O)— in which thecarbonyl is attached to the accm.

The invention also describes kits for detecting or determining amolecule or molecules of the JWH family and/or CP family comprising oneor more antibodies of the invention. Preferably, the kit comprises oneor more antibodies raised to an immunogen of either structure (a), (b),(c) or (d) of Group I and one or more antibodies raised to an immunogenof structure (d), (e), (f) or (g) of Group II. More preferably theantibodies of the kit are derived from an immunogen of structure (a)and/or (c). A kit comprising antibodies derived from Immunogens I or II(FIG. 5) for detecting or determining one or more of JWH-018, JWH-073,JWH-200 and JWH-398 is particularly preferred. Optionally, the kit maycomprise antibodies derived from Immunogens I or II for additionally oralternatively detecting or determining one of more of JWH-081, JWH-018N-pentanoic acid metabolite (M1), JWH-018 5-hydroxyindole metabolite(M2), JWH-018 4-hydroxyindole metabolite (M3), JWH-018N-(5-hydroxypentyl) metabolite (M4), JWH-018 6-hydroxyindole metabolite(M5), JWH-018 N-(4-hydroxypentyl) metabolite,1-(5-fluoropentyl)indol-3-yl(1-naphthyl) methanone, JWH-250, JWH-073N-(3-hydroxybutyl) metabolite and JWH-073 N-(4-hydroxybutyl) metabolite.

The antibodies of the kit are preferably tethered to any suitable solidsupport such as a chip. Although the solid support can be of anysuitable shape such as a bead or a slide and of any suitable materialsuch as silicon, glass or plastic, the solid support is preferably aceramic chip. The kit may further include calibrators and one or moredetecting agents and optionally includes instructions for the use of theantibodies of the kit and if incorporated, the calibrators and detectingagents, for detecting and determining molecules from the JWH and/or CPfamilies. The invention also embodies solid supports comprising thenovel antibodies.

The antibodies of the invention are used for the detection ordetermination of JWH and/or CP molecules either in herbal mixtures, anin vitro sample taken from an individual or any other substancesuspected of their incorporation. A preferred use of the antibodies ofthe invention is their use in the detection and/or quantification ofJWH-018, JWH-073, JWH-200 and JWH-398 in herbal mixtures and/or JWH-073,JWH-200, JWH-398 and JWH-018 and its metabolites in in vitro samplestaken from individuals.

General Methods, Examples and Results Preparation of Haptens, Immunogensand Detecting Agents

Although haptens provide defined structural epitopes, they are not inthemselves immunogenic and therefore need to be conjugated to carriermaterials, which will elicit an immunogenic response when administeredto a host animal. Appropriate carrier materials commonly containpoly(amino acid) segments and include polypeptides, proteins and proteinfragments. Illustrative examples of useful carrier materials are bovineserum albumin (BSA), egg ovalbumin, bovine gamma globulin, bovinethyroglobulin (BTG), keyhole limpet haemocyanin (KLH) etc.Alternatively, synthetic poly(amino acids) having a sufficient number ofavailable amino groups, such as lysine, may be employed, as may othersynthetic or natural polymeric materials bearing reactive functionalgroups. Also, carbohydrates, yeasts or polysaccharides may be conjugatedto the hapten to produce an immunogen. The haptens can also be coupledto a detectable labelling agent such as an enzyme (for example,horseradish peroxidase), a substance having fluorescent properties or aradioactive label for the preparation of detecting agents for use in theimmunoassays. The fluorescent substance may be, for example, amonovalent residue of fluorescein or a derivative thereof. Immunogenformation for the invention described herein involves conventionalconjugation chemistry. In order to confirm that adequate conjugation ofhapten to carrier material has been achieved, prior to immunisation,each immunogen is evaluated using matrix-assisted UV laserdesorption/ionisation time-of-flight mass spectroscopy (MALDI-TOF MS).

General Procedure for MALDI-TOF Analysis of Immunogens

MALDI-TOF mass spectrometry was performed using a Voyager STRBiospectrometry Research Station laser-desorption mass spectrometercoupled with delayed extraction. An aliquot of each sample to beanalysed was diluted in 0.1% aqueous trifluoroacetic acid (TFA) tocreate 1 mg/ml sample solutions. Aliquots (1 μl) were analysed using amatrix of sinapinic acid and bovine serum albumin (Fluka) was used as anexternal calibrant.

Preparation of Antisera

In order to generate polyclonal antisera, an immunogen of the presentinvention is mixed with Freund's adjuvant and the mixture is injectedinto a host animal, such as rabbit, sheep, mouse, guinea pig or horse.Sheep are the preferred host animal. Further injections (boosts) aremade and serum is sampled for evaluation of the antibody titre. When theoptimal titre has been attained, the host animal is bled to yield asuitable volume of specific antiserum. The degree of antibodypurification required depends on the intended application. For manypurposes, there is no requirement for purification, however, in othercases, such as where the antibody is to be immobilised on a solidsupport, purification steps can be taken to remove undesired materialand eliminate non-specific binding.

Immunoassay Development

The process of developing an immunoassay is well known to the personskilled in the art. Briefly, for a competitive immunoassay in which thetarget analyte is a non-immunogenic molecule such as a hapten, thefollowing process is conducted: antibodies are produced by immunising ananimal, preferably a mammalian animal, by repeated administration of animmunogen. The serum from the immunised animal is collected when theantibody titre is sufficiently high. A detecting agent is added to asample containing the target analyte and the raised antibodies, and thedetecting agent and analyte compete for binding to the antibodies. Theprocess may comprise fixing said serum antibodies to a backing substratesuch as a polystyrene solid support or a ceramic chip. The antibodiescan be polyclonal or monoclonal using standard techniques. The signalemitted in the immunoassay is proportionate to the amount of detectingagent bound to the antibodies which in turn is inversely proportionateto the analyte concentration. The signal can be detected or quantifiedby comparison with a calibrator.

EXAMPLES Example 1: Preparation of 3-(1-naphthoyl)-1H-indole 1

To a cooled solution of indole (5.85 g, 50 mmol) in ether (50 ml) undernitrogen was added slowly a solution of methylmagnesium bromide (3M) inether (17.5 ml). After addition, the reaction mixture was warmed up toroom temperature and stirred for 2 h at room temperature. Then themixture was cooled down again to 0° C., and to it was added slowly withstirring a solution of 1-naphthoyl chloride (9.5 g, 50 mmol) in ether(50 ml). The resulting mixture was warmed up to room temperature andstirred for 2 h at room temperature followed by slow addition ofsaturated ammonium chloride solution (375 ml). The mixture was thenstirred overnight at room temperature. A white solid was formed,filtered, washed by ether and dried under high vacuum to give3-(1-naphthoyl)-1H-Indole 1 (12.3 g, 91%).

Example 2: Preparation ofN-(5-Ethoxycarbonylpentyl)-3-(1-naphthoyl)-1H-indole 2

To a suspension of sodium hydride (1.1 g, 30 mmol, 60% in mineral oil)in DMF (100 ml) under nitrogen was added solid 3-(1-naphthoyl)-1H-indole1 (5.43 g, 20 mmol). After stirring at room temperature for 1 h, asolution of ethyl 6-bromohexanoate (6.6 g, 30 mmol) in DMF (10 ml) wasadded slowly with stirring over a period of 15 min and the mixture wasthen heated at 60° C. for 3 h. The solvent was removed under high vacuumand the crude product was suspended in water (150 ml) and extracted byethyl acetate (2×150 ml). The combined ethyl acetate phases were washedby water (1×100 ml), brine (1×100 ml), dried over sodium sulphatefiltered and concentrated to dryness. The crude product obtained waspurified by chromatography on silica gel using hexane/ethyl acetate(8/2) to give the title compound 2 as an oil which became solid in thecold (7.1 g, 86%).

Example 3: Preparation of N-(5-carboxypentyl)-3-(1-naphthoyl)-1H-indole(Hapten-A)

To a solution of 2 (5.0 g, 12 mmol) in a mixture of THF/H₂O (1:1) wasadded potassium hydroxide (1.7 g) and the mixture was stirred at 60° C.for 1 h. The THF was removed under vacuum, the aqueous solutionacidified to pH 1 by the addition of hydrochloric acid solution (1N) andextracted by ethyl acetate (3×100 ml). The combined organic phases werewashed by water (100 ml), brine solution (100 ml), dried over sodiumsulphate, filtered and concentrated to dryness. The crude productobtained was dissolved in ethyl acetate (10 ml) and the Hapten-Aprecipitated by the addition of a mixture of ether/hexane as a whitesolid, filtered and dried under high vacuum to give Hapten-A (3.6 g,78%).

Example 4: Conjugation of Hapten-A to BSA

To a solution of Hapten-A (52.2 mg, 0.13 mmol) in DMF (1.0 ml) was addedN,N-dicyclohexylcarbodiimide (DCC) (34.0 mg, 0.16 mmol) andN-hydroxysuccinimide (19.0 mg, 0.16 mmol) and the mixture was stirred atroom temperature overnight. The dicyclohexylurea formed was removed byfiltration and the solution added dropwise to a solution of BSA (200 mg,3.0 μmol) in 50 mM sodium bicarbonate solution (pH 8.5) (10 ml). Themixture was then stirred overnight at 4° C. The solution was thendialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 h at4° C., and freeze-dried. MALDI results showed 9.83 molecule of Hapten-Ahad been conjugated to one molecule of BSA.

Example 5: Conjugation of Hapten-A to BTG (Immunogen I)

To a solution of Hapten-A (58.0 mg, 0.15 mmol) in DMF (1.0 ml) was addedN,N-dicyclohexylcarbodiimide (DCC) (34.0 mg, 0.165 mmol) andN-hydroxysuccinimide (19.0 mg, 0.16 mmol) and the mixture was stirred atroom temperature overnight. The dicyclohexylurea formed was removed byfiltration and the solution was added dropwise to a solution of BTG (150mg) in 50 mM sodium bicarbonate solution (pH 8.5) (10 ml). The mixturewas then stirred overnight at 4° C. The solution was then dialysedagainst 50 mM phosphate buffer pH 7.2 (3 changes) for 24 h at 4° C., andfreeze-dried to give Immunogen I.

Example 6: Conjugation of Hapten-A to HRP

EDC hydrochloride (10 mg) was dissolved in water (0.5 ml) andimmediately added to a solution of Hapten-A (2 mg) in DMF (0.2 ml).After mixing, this solution was added dropwise to a solution of HRP (20mg) in water (1 ml). Sulfo-NHS (5 mg) was added and the reaction mixturewas incubated in the dark at room temperature overnight. Excess haptenwas removed with double PD-10 columns (Pharmacia) in series,pre-equilibrated with PBS at pH 7.2. The hapten-HRP detecting agent wasthen dialysed overnight against 10 L of PBS at pH 7.2 at 4° C.

Example 7: Preparation of 5-methoxy-3-(1-naphthoyl)-1H-indole 4

To a cooled solution at 0° C. of 5-methoxy-1H-indole 3 (7.4 g, 50 mmol)in diethyl ether (150 ml) under nitrogen was added dropwise a solutionof methylmagnesium bromide (3M) in diethyl ether (17.5 ml) and themixture was stirred for 2 h at room temperature. The solution was thencooled at 0° C. and to this solution was added a solution of1-naphthoylchloride (9.5 g, 50 mmol) in diethyl ether (100 ml) dropwiseover a period of 15 min. After the addition was completed the solutionwas warmed up at room temperature and stirred for 2 h followed by slowaddition of saturated ammonium chloride solution (375 ml) and stirredovernight. The white solid formed was filtered, washed by ether anddried under high vacuum to give 5-methoxy-3-(1-naphthoyl)-1H-indole 4(11.3 g, 75%).

Example 8: Preparation of 5-methoxy-3-(1-naphthoyl)-N-pentyl-1H-indole 5

To a suspension of sodium hydride (1.54 g, 45.7 mmol, 60% in mineraloil) in DMF (100 ml) under nitrogen was added dropwise a solution of5-methoxy-3-(1-naphthoyl)-1H-indole 4 (9.83 g, 32.6 mmol) in DMF (50 ml)and the mixture was stirred at 40° C. for 1 h. The solution was thencooled to room temperature and to this mixture was added a solution of1-bromopentane (8.2 g, 54.3 mmol) in DMF (25 ml). The mixture wasstirred at 60° C. for 1 h. The solvent was removed under high vacuum andthe crude product was suspended in water (200 ml) and extracted by ethylacetate (2×200 ml). The combined ethyl acetate phases were washed bywater (1×100 ml), brine (1×100 ml), dried over sodium sulphate, filteredand concentrated to dryness. The crude product obtained was purified bychromatography on silica gel using hexane/ethyl acetate (7/3) to givethe title compound 5 as an oil which solidified upon cooling (7.1 g,59%).

Example 9: Preparation of 5-hydroxy-3-(1-naphthoyl)-N-pentyl-1H-indole 6

To a solution of hydrobromic acid (48 w/w/%) in water (150 ml) was added5 (6.5 g, 17.5 mmol) and the mixture was heated at reflux for 3 h. Thesolution was cooled to room temperature and concentrated to dryness.Water was then added (200 ml), the solution neutralized to pH 7-8 andthen extracted with ethyl acetate (3×150 ml). The combined organiclayers were washed by water (150 ml), brine (150 ml), dried over Na₂SO₄,filtered and concentrated to dryness. The crude product was thenrecrystallized from ethyl acetate/hexane to give a white solid of5-hydroxy-3-(1-naphthoyl)-N-pentyl-1H-indole 6 (4.5 g, 72%).

Example 10: Preparation of5-(tert-butoxycarbonylmethoxy)-3-(1-naphthoyl)-N-pentyl-1H-indole 7

To a suspension of sodium hydride (452 mg, 13.4 mmol, 60% in mineraloil) in DMF (25 ml) under nitrogen was added dropwise a solution of 6(3.7 g, 10.35 mmol) in DMF (50 ml) and the mixture was stirred at 60 Cfor 1 h. The solution was then cooled to room temperature and to thismixture was added a solution of tert-butyl bromoacetate (2.62 g, 13.4mmol) in DMF (25 ml). The mixture was stirred at 60° C. for 3 h. The DMFwas removed under high vacuum and the crude product was suspended inwater (100 ml) and the mixture was extracted by ethyl acetate (2×100ml). The combined ethyl acetate phases were washed by water (1×50 ml),brine (1×50 ml), dried over sodium sulphate filtered and concentrated todryness. The crude product obtained was purified by chromatography onsilica gel using hexane/ethyl acetate (9/1) to give the title compound 7(3.5 g, 72%).

Example 11: Preparation of5-carboxymethoxy-3-(1-naphthoyl)-N-pentyl-1H-indole (Hapten-B)

To a solution of 7 (3.0 g, 6.4 mmol) in dichloromethane (50 ml) wasadded TFA (25 ml) and the mixture was stirred at room temperature for 3h. The mixture was evaporated to dryness and the crude obtained waspurified by chromatography on silica-gel using 5% methanol in chloroformto give Hapten-B (2.1 g, 79%). NMR ¹³C (CD₃OD, 5 ppm): 194.55, 173.21,156.53, 141.14, 140.15, 135.25, 134.10, 131.98, 131.17, 129.46, 128.9,1127.85, 127.44, 126.87, 125.55, 125.85, 117.92, 115.14, 112.72, 106.33,65.54, 30.59, 29.87, 23.2, 14.25.

Example 12: Conjugation of Hapten-B to BSA

To a solution of Hapten-B (62.4 mg, 0.15 mmol) DMF (1.0 ml) was addedN,N-dicyclohexylcarbodiimide (DCC) (34.1 mg, 0.16 mmol) andN-hydroxysuccinimide (19.02 mg, 0.16 mmol) and the mixture was stirredat room temperature overnight. The dicyclohexylurea formed was removedby filtration and the solution was added dropwise to a solution of BSA(200 mg, 3 μmol) in 50 mM sodium bicarbonate solution (pH 8.5) (10 ml).The mixture was then stirred overnight at 4 C. The solution was thendialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 h at 4C, and freeze-dried. MALDI results showed 37.2 molecules of Hapten-B hadbeen conjugated to one molecule of BSA.

Example 13: Conjugation of Hapten-B to BTG (Immunogen II)

To a solution of Hapten-B (56.0 mg, 0.13 mmol) in DMF (1.0 ml) was addedN,N-dicyclohexylcarbodiimide (DCC) (30.6 mg, 0.14 mmol) andN-hydroxysuccinimide (17.1 mg, 0.14 mmol) and the mixture was stirred atroom temperature overnight. The dicyclohexylurea formed was removed byfiltration and the solution was added dropwise to a solution of BTG (150mg) in 50 mM sodium bicarbonate solution (pH 8.5) (15 ml). The mixturewas then stirred overnight at 4° C. The solution was then dialysedagainst 50 mM phosphate buffer pH 7.2 (3 changes) for 24 h at 4° C., andfreeze-dried to give Immunogen II.

Example 14: Preparation of 3-carboxy-1-pentyl-1H-indole (Hapten-C)

Indole 3-carboxylic acid (3 g, 18.62 mmol) was added to a suspension ofsodium hydride (60% in oil) (1.1 lg, 1.5 eq) in dry DMF (30 ml) under anitrogen atmosphere. The mixture was stirred at room temperature for 45min (H₂ evolving has ceased) and to this was added 1-bromopentane (4.62ml, 2 eq) in dry DMF (10 ml) dropwise. The mixture was stirred at roomtemperature overnight. The solvents were removed in vacuo and to theresidue was added water (30 ml) and ethyl acetate (30 ml). The ethylacetate portion was separated, dried over sodium sulphate, filtered andevaporated to dryness. The crude residue was purified by columnchromatography (silica gel: 20% ethyl acetate in hexane) to give thetitle compound (2.12 g, 49%) as a cream solid.

Example 15: Conjugation of 3-carboxy-1-pentyl-1H-indole (Hapten C) toBSA

To a solution of Hapten-C (26.13 mg, 0.113 mmol) in DMF (1.0 ml) wasadded N,N-dicyclohexylcarbodiimide (DCC) (25.64, 0.1243 mmol) andN-hydroxysuccinimide (14.30 mg, 0.1243 mmol) and the mixture was stirredat room temperature overnight. The dicyclohexylurea formed was removedby filtration and the solution was added dropwise to a solution of BSA(150 mg) in 100 mM sodium bicarbonate solution (pH 8.5) (9 ml). Themixture was then stirred overnight at 4° C. The solution was thendialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hoursat 4° C., and freeze-dried.

Example 16: Conjugation of 1-pentyl-1H-indole-3-carboxylic acid(3-carboxy-1-pentyl-1H-indole, Hapten C) to BTG (Immunogen III)

To a solution of Hapten-C (31.22 mg, 0.135 mmol) in DMF (1.0 ml) wasadded N,N-dicyclohexylcarbodiimide (DCC) (30.74 mg, 0.149 mmol) andN-hydroxysuccinimide (17.1 mg, 0.149 mmol) and the mixture was stirredat room temperature overnight. The dicyclohexylurea formed was removedby filtration and the solution was added dropwise to a solution of BTG(150 mg) in 100 mM sodium bicarbonate solution (pH 8.5) (15 ml). Themixture was then stirred overnight at 4° C. The solution was thendialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hoursat 4° C., and freeze-dried to give Immunogen III.

Example 17: Conjugation of Hapten-B to HRP

EDC hydrochloride (10 mg) was dissolved in water (0.5 ml) andimmediately added to a solution of Hapten-B (2 mg) in DMF (0.2 ml).After mixing, this solution was added dropwise to a solution of HRP (20mg) in water (1 ml). Sulfo-NHS (5 mg) was added and the reaction mixturewas incubated in the dark at room temperature overnight. Excess haptenwas removed with double PD-10 columns (Pharmacia) in series,pre-equilibrated with PBS at pH 7.2. The hapten-HRP detecting agent wasthen dialysed overnight against 10 L of PBS at pH 7.2 at 4° C.

Example 18: Preparation of Antibodies to Immunogens I and II

Aqueous solutions of the immunogens prepared in examples 5 and 13 wereformulated with Freund's Complete Adjuvant (FCA) to form emulsionsconsisting of 2 mg/ml Immunogen I and 2 mg/ml of Immunogen II in 50%(v/v) FCA. Three sheep were immunised with each emulsion (1°immunisations), 0.25 ml being intramuscularly injected at four sites inthe rump of each animal. Subsequent immunisations (boosts 2-8) contained1 mg/ml Immunogen I and 1 mg/ml Immunogen II. All boosts were emulsifiedin 50% (v/v) Freund's Incomplete Adjuvant (FIA) and administered to theappropriate sheep in the same manner as the 1° immunisations, at monthlyintervals. Blood sampling took place 7-14 days after each boost. Eachsample was processed to produce antiserum, which was further purified bycaprylic acid and ammonium sulphate precipitation to yield animmunoglobulin (Ig) fraction. The Ig fraction was evaluated bycompetitive ELISA microtiter plate assay, as described in example 19below.

Example 19: Development of Competitive ELISA for JWH SyntheticCannabinoids and Metabolites

(a) The wells of an enhanced binding 96 well polystyrene microtiterplate were coated with the Ig fraction of the antiserum raised toImmunogen II (Hapten B-BTG—example 13) and diluted in 10 mM Tris, pH8.5(1250/well). The appropriate antibody coating concentration wasdetermined using standard ELISA checkerboard techniques. The plate wasincubated for 2 hours at 37° C., washed 4 times over a 10-minute periodwith Tris buffered saline containing Tween 20 (TBST) and tapped dry.Standard solutions of JWH synthetic cannabinoids, metabolites andselected molecules were prepared in TBST and 50 μl of each was added tothe appropriate wells. 750 of conjugate (Hapten A-HRP) diluted in Trisbuffer containing EDTA, D-mannitol, sucrose, thimerosal and BSA, wasadded to each of the wells. The appropriate dilution of conjugate wasalso determined using standard ELISA checkerboard techniques. The platewas incubated at 25° C. for 1 hour. The excess unbound conjugate wasremoved by washing 6 times over a 10-minute period with TBST and tappeddry. 125 μl of tetramethylbenzedine (TMB) substrate solution was addedto each well of the plate that was then incubated for 15-20 minutes inthe dark at room temperature. The reaction was terminated by addition of125 μl of 0.2M sulphuric acid to each well. The absorbance was thenmeasured at 450 nm using a microtiter plate reader.

Employing each series of standards, calibration curves were generatedand these were used to determine the specificity of the immunoassay forthe JWH synthetic cannabinoids, metabolites and selected molecules. Theresults of this study are presented in Tables 1 to 3, cross-reactivitybeing calculated according to the following formula:% CR=IC ₅₀,_(JWH-018) /IC _(50,CR)×100

Where % CR is the percentage cross-reactivity, IC_(50, JWH-018) is theconcentration of JWH-018 that causes 50% displacement of signal andIC50, CR is the concentration of JWH syntheticcannabinoid/metabolite/selected molecule that causes 50% displacement ofsignal.

Table 1: Data generated from a competitive microtiter plate assay forJWH synthetic cannabinoids and metabolites, employing antiserum raisedto Immunogen II (Hapten B-BTG) and conjugate (Hapten A-HRP) as detectionreagent.

(b) In a similar manner to that described in Example 19(a), the wells ofa 96 well microtiter plate were coated with the Ig fraction of theantiserum raised to Immunogen I (Hapten A-BTG—example 5). Conjugate(Hapten A-HRP) was employed as a detection reagent. The data generatedis presented in Table 2.

Table 2: Data generated from a competitive microtiter plate assay forJWH synthetic cannabinoids and metabolites, employing antiserum raisedto Immunogen I (Hapten A-BTG) and conjugate (Hapten A-HRP) as detectionreagent.

(c) The resulting competitive ELISA for synthetic cannabinoids andmetabolites was farther employed to analyse urine and serum samples from20 patients (Table 3).

(d) The resulting competitive ELISA for synthetic cannabinoids andmetabolites was also employed to test for the 5-fluoropentyl derivativeof JWH-018, (1-(5-fluoropentyl)indol-3yl (1-naphthyl) methanone (Table4).

Results

TABLE 1 Antibody characterisation using antiserum raised to Immunogen IIand detecting agent derived from Hapten-A in a competitive assay format(CR based on 100% for JWH-018) Analyte IC₅₀ ng/ml % Cross-reactivityJWH-018 2.11 100.00 JWH-073 1.56 135.26 JWH-398 17.55  12.02 JWH-2001.66 127.11 3-(1-naphthoyl)-1H-Indole >>40     <<5.28 M1 5.48 38.50 M21.62 130.25 M3 9.16 23.03 M4 1.15 183.48 M5 0.98 215.31 >>implies avalue greatly exceeding the value given (40 ng/ml was the highestconcentration tested) <<implies a value greatly below the value given

TABLE 2 Antibody characterisation using antiserum raised to Immunogen Iand detecting agent derived from Hapten-A in a competitive assay format(CR based on 100% for JWH-018) Analyte IC₅₀ ng/ml % Cross-reactivityJWH-018 2.71 100.00 JWH-073 0.93 291.40 JWH-398 >>40     <<6.78 JWH-2000.31 874.19 3-(1-naphthoyl)-1H-Indole 3.84 70.57 Ml 0.42 645.24 M2 0.39694.87 M3 25.51  10.62 M4 0.18 1505.56 M5 2.04 132.84 >>implies a valuegreatly exceeding the value given (40 ng/ml was the highestconcentration tested) <<implies a value greatly below the value given

TABLE 3 Sensitivity and cross-reactivity (CR) of antibodies raised toImmunogens I & II of selected molecules Standard Conc^(n) Analyte ng/mlIC₅₀ ng/ml % CR Serotonin 750.00 >750.00 <0.28 4-Methoxypsilocin750.00 >750.00 <0.28 Delta-9-THC 750.00 >750.00 <0.28 Cannabinol750.00 >750.00 <0.28 11-Hydroxy-δ-9-THC 750.00 >750.00 <0.28 CP 47,497750.00 >750.00 <0.28 3-Carboxy-N-pentyl-1H-indole 750.00 >750.00 <0.283-Carboxy-1H-indole 750.00 >750.00 <0.28 3-Carboxymethyl-5-hydroxy-1H-750.00 >750.00 <0.28 indole 5-Hydroxytryptophol 750.00 >750.00 <0.28

TABLE 4 Sensitivity and cross-reactivity (CR) of antibodies raised toImmunogens I & II of JWH-018 and 1-(5-Fluoropentyl)indol- 3-yl(1-naphthyl) methanone* (5-Fluoropentyl derivative) Immunogen IC₅₀ ng/ml% Cross-reactivity I JWH-018 2.30 100.00 I 5-Fluoropentyl derivative0.02 11500.00 II JWH-018 1.97 100.00 II 5-Fluoropentyl derivative 0.22908.00 *Binding of this molecule to the CB1 receptor is detailed in U.S.Pat. No. 6,900,236

Immunoassays using antibodies of the invention to test for potentialcross-reactants (Table 3) and to screen the urine and serum of twentypatients for cross-reactive molecules did not reveal any cross-reactantswhich could invalidate the measurements taken using the antibodies,methods, kits and products of the invention.

As can be seen from Tables 1 to 3, for the first time antibodies havebeen provided that bind to various JWH molecules, metabolites andpotential metabolites, whereas other common indole-containing moleculesand non-JWH CB1-active molecules do not bind. The antibody produced fromImmunogen I is able to bind to JWH molecules known to be incorporated inherbal therapeutics such as JWH-018, proposed metabolites such as M2(Wintermeyer et al 2010), and potential metabolites such as M5. Thedetection and quantification of other JWH SSCs is also provided for, theantibodies of the invention bind a range of molecules comprising the3-(1-naphthoyl)-1H-indole structure (e.g. JWH-071, JWH-398, M1 etc.).Table 3 confirms that the same antibodies do not cross-react with otherpsychoactive drugs, with molecules present in biological samples ofpatients who have not taken JWH containing substances or with moleculeswithout the 3-(1-naphthoyl)-1H-indole such as 3-carboxy-1H-indole. Theconcept of using the antibodies of the invention to detect and determinefuture stealth synthetic cannabinoids is highlighted in Table 4; the5-fluoropentyl derivative known to bind the CBI receptor, but which asof yet has not been detected in herbal therapeutics or similarsubstances, binds to antibodies raised from Immunogens I and II.

The invention claimed is:
 1. A polyclonal antibody raised to animmunogen of structure

wherein “BTG” is bovine thyroglobulin, wherein the polyclonal antibodyhas a cross-reactivity of 100% for JWH-018, wherein the polyclonalantibody has a cross-reactivity of at least 100% for JWH-073, JWH-200,JWH-018 6-hydroxyindole metabolite (M5), JWH-018 N-(5-hydroxypentyl)metabolite (M4), and JWH-018 5-hydroxyindole metabolite (M2), whereinthe polyclonal antibody has a cross-reactivity of less than 0.28%, basedon 100% cross-reactivity for JWH-018, for each molecule selected fromthe group consisting of serotonin, 4-methoxypsilocin, delta-9-THC,cannabinol, 11-hydroxy-δ-9-THC, CP 47,497, 3-carboxy-N-pentyl-1H-indole,3-carboxy-1H-indole, 3-carboxymethyl-5-hydroxy-1H-indole and5-hydroxytryptophol, and wherein the polyclonal antibody has a crossreactivity related to JWH-018 of less than 5.28% for3-(1-naphthoyl)-1H-indole, using a detecting agent derived from HaptenA:

conjugated at the carboxy group to horseradish peroxidase (HRP).
 2. Thepolyclonal antibody of claim 1, wherein the polyclonal antibody binds toat least one molecule selected from the group consisting of JWH-398,JWH-018 N-pentanoic acid metabolite (M1), JWH-018 4-hydroxyindolemetabolite (M3), and 1-(5-fluoropentyl)indol-3-yl (1-naphthyl)methanone.
 3. The polyclonal antibody of claim 1, having an IC₅₀ ofgreater than 750 ng/ml for each molecule selected from the groupconsisting of serotonin, 4-methoxypsilocin, delta-9-THC, cannabinol,11-hydroxy-δ-9-THC, CP 47,497, 3-carboxy-N-pentyl-1H-indole,3-carboxy-1H-indole, 3-carboxymethyl-5-hydroxy-1H-indole and5-hydroxytryptophol.
 4. A method of detecting or determining at leastone synthetic cannabinoid of the JWH family, a metabolite thereof or anycombinations thereof in an in vitro sample of an individual or in asolution derived from a substance suspected of containing syntheticcannabinoids, the method comprising contacting the sample or solutionwith at least one detecting agent and the polyclonal antibody of claim1; detecting or determining the quantity of the at least one detectingagent; and deducing from calibrators the presence of or amount of the atleast one synthetic cannabinoid of the JWH family, a metabolite thereofor any combinations thereof in the sample or solution.
 5. The method ofclaim 4, wherein the synthetic cannabinoids to be detected or determinedare selected from the group consisting of JWH-018, JWH-073, JWH-200 andJWH-398.
 6. A kit for detecting or determining at least one molecule ofthe JWH family, a metabolite thereof or any combinations thereof, thekit comprising the polyclonal antibody of claim
 1. 7. The kit of claim6, wherein the at least one molecule of the JWH family, a metabolitethereof or a combination thereof is selected from the group consistingof JWH-018, JWH-073, JWH-200, JWH-398, JWH-018 N-pentanoic acidmetabolite (M1), JWH-018 5-hydroxyindole metabolite (M2), JWH-0184-hydroxyindole metabolite (M3), JWH-018 N-(5-hydroxypentyl) metabolite(M4), JWH-018 6-hydroxyindole metabolite (M5), JWH-018N-(4-hydroxypentyl) metabolite, 1-(5-fluoropentyl)indol-3-yl(1-naphthyl) methanone, JWH-073 N-(3-hydroxybutyl) metabolite andJWH-073 N-(4-hydroxybutyl) metabolite.
 8. The kit of claim 7, whereinthe at least one molecule of the JWH family is selected from the groupconsisting of JWH-018, JWH-073, JWH-200 and JWH-398.
 9. The kit of claim6, wherein the polyclonal antibody has an IC₅₀ of greater than 750 ng/mlfor each molecule selected from the group consisting of serotonin,4-methoxypsilocin, delta-9-THC, cannabinol, 11-hydroxy-δ-9-THC, CP47,497, 3-carboxy-N-pentyl-1H-indole, 3-carboxy-1H-indole,3-carboxymethyl-5-hydroxy-1H-indole and 5-hydroxytryptophol.
 10. The kitof claim 6, wherein the polyclonal antibody binds to each one selectedfrom the group consisting of JWH-073, JWH-200, JWH-018, JWH-018N-pentanoic acid metabolite (M1), JWH-018 5-hydroxyindole metabolite(M2), JWH-018 4-hydroxyindole metabolite (M3), JWH-018N-(5-hydroxypentyl) metabolite (M4) and JWH-018 6-hydroxyindolemetabolite (M5).